Supplement of Biogeosciences, 14, 1527–1539, 2017
http://www.biogeosciences.net/14/1527/2017/
doi:10.5194/bg-14-1527-2017-supplement
© Author(s) 2017. CC Attribution 3.0 License.
Supplement of
New insights on resource stoichiometry: assessing availability of carbon,
nitrogen, and phosphorus to bacterioplankton
Ana R. A. Soares et al.
Correspondence to: Ana R. A. Soares ([email protected])
The copyright of individual parts of the supplement might differ from the CC-BY 3.0 licence.
Table 1. Schematic representation of the standard growth curves design
Experiment Step
Sampling, pre-filtration,
storage
Sample division
Filtration
and
Inoculation, addition of “L16”
Induction of element limitation
Subsample division
Bioassay “spiking” with known
element concentration
Standard C curve
Standard N curve
Standard P curve
Sub-volume 1
0.2 μm filtration
2 % (v/v) standard inoculum
5 % (v/v) “L16” bacterial growth medium
C-limitation induced
+2000 μg N L-1 as NH4NO3
+200 μg P L-1 as Na2HPO4
subsubsubsubsubvolume
volume
volume
volume
volume
1.1
1.2
1.3
1.4
1.5
+330
+660
+1000
+1330
+1500
μg C L-1 μg C L-1 μg C L-1 μg C L-1 μg C L-1
as
as
as
as
as
C6H12O6 C6H12O6 C6H12O6 C6H12O6 C6H12O6
(C1)
(C2)
(C3)
(C4)
(C5)
Water sampled September 2012
0.7 μm filtration, storage at 1 °C
Sub-volume 2
0.2 μm filtration
2 % (v/v) standard inoculum
5 % (v/v) “L16” bacterial growth medium
N-limitation induced
+20000 μg C L-1 as C6H12O6
+200 μg P L-1 as Na2HPO4
subsubsubsubsubvolume
volume
volume
volume
volume
2.1
2.2
2.3
2.4
2.5
+105
+133
+ 205
+305
+405
μg N L-1 μg N L-1 μg N L-1
μg N L-1 μg N L-1
as
as
as
as
as
NH4NO3 NH4NO3 NH4NO3 NH4NO3 NH4NO3
(N1)
(N2)
(N3)
(N4)
(N5)
Sub-volume 3
0.2 μm filtration
2 % (v/v) standard inoculum
5 % (v/v) “L16” bacterial growth medium
P-limitation induced
+20000 μg C L-1 as C6H12O6
+2000 μg N L-1 as NH4NO3
subsubsubsubsubvolume
volume
volume
volume
volume
3.1
3.2
3.3
3.4
3.5
+15.5
+18.8
+20.5
+30.5
+40.5
μg P L-1
μg P L-1
μg P L-1
μg P L-1
μg P L-1
as
as
as
as
as
Na2HPO4 Na2HPO4 Na2HPO4 Na2HPO4 Na2HPO4
(P1)
(P2)
(P3)
(P4)
(P5)
Standard growth curves
(μg C L-1)
(nmol leucine L-1)
(nmol leucine L-1)
C1 C2 C3 C4 C5
N1 N2 N3 N4 N5
(μg N L-1)
C slope
N slope
(nmol leucine L-1 per μg C L-1)
(nmol leucine L-1 per μg N L-1)
(nmol leucine L-1)
7-day incubation at 20 °C in Eppendorf tubes
Measurements of leucine incorporation at discrete time
points (t1, t2, t2, t3, t4, t7) and integration for 7 days
Leucine incorporation
7-day incubation at 20 °C in Eppendorf tubes
Measurements of leucine incorporation at discrete time
points (t1, t2, t2, t3, t4, t7) and integration for 7 days
Leucine incorporation
7-day incubation at 20 °C in Eppendorf tubes
Measurements of leucine incorporation at discrete time
points (t1, t2, t2, t3, t4, t7) and integration for 7 days
Leucine incorporation
Bioassay incubation
Determination and integration
of leucine uptake
P1
P2 P3 P4 P5
(μg P L-1)
P slope
(nmol leucine L-1 per μg P L-1)
Table 2. Schematic representation of experimental controls design
Experiment step
C control
N control
P control
Inoculation
Addition of “L16”
2 % (v/v) standard inoculum
5 % (v/v) “L16” bacterial growth medium
C-limitation induced
+2000 μg N L-1 as NH4NO3
+200 μg P L-1 as Na2HPO4
7-day incubation at 20 °C in Eppendorf tubes
(5 replicates)
Measurements of leucine incorporation at discrete
time points (t1, t2, t2, t3, t4, t7) and
integration for 7 days
2 % (v/v) standard inoculum
5 % (v/v) “L16” bacterial growth medium
N-limitation induced
+20000 μg C L-1 as C6H12O6
+200 μg P L-1 as Na2HPO4
7-day incubation at 20 °C in Eppendorf tubes
(5 replicates)
Measurements of leucine incorporation at discrete
time points (t1, t2, t2, t3, t4, t7) and
integration for 7 days
2 % (v/v) standard inoculum
5 % (v/v) “L16” bacterial growth medium
P-limitation induced
+20000 μg C L-1 as C6H12O6
+2000 μg N L-1 as NH4NO3
7-day incubation at 20 °C in Eppendorf tubes
(5 replicates)
Measurements of leucine incorporation at discrete
time points (t1, t2, t2, t3, t4, t7) and
integration for 7 days
Induction of element limitation
Bioassay incubation
Determination and integration of leucine
uptake
Table 3. Schematic representation of determination of bioavailable C, N and P
Experiment step
Bioavailable C
Sampling, pre-filtration, storage
Sample division
Filtration
and
Inoculation, addition of “L16”
Induction of element limitation
Bioassay incubation
Determination and integration of leucine uptake
Correction for blanks
Determination of amounts of bioavailable
element taken up during 7 days
Sub-volume 1
0.2 μm filtration
2 % (v/v) standard inoculum
5 % (v/v) “L16” bacterial growth medium
C-limitation induced
+2000 μg N L-1 as NH4NO3
+200 μg P L-1 as Na2HPO4
7-day incubation at 20 °C in Eppendorf tubes
(5 replicates)
Measurements of leucine incorporation at discrete
time points (t1, t2, t2, t3, t4, t7) and integration
for 7 days
Corrected leucine incorporation = average
measurements of leucine incorporation (n = 5) –
average leucine incorporation from experimental
controls (n = 5)
Bioavailable C taken up (μg C L-1)=
Corrected leucine incorporation
(nmol of leucine L-1)
÷
C slope (nmol leucine L-1 per μg C L-1)
Bioavailable N
Water sample,
0.7 μm filtration
1 °C storage
Sub-volume 2
0.2 μm filtration
2 % (v/v) standard inoculum
5 % (v/v) “L16” bacterial growth medium
N-limitation induced
+20000 μg C L-1 as C6H12O6
+200 μg P L-1 as Na2HPO4
7-day incubation at 20 °C in Eppendorf tubes
(5 replicates)
Measurements of leucine incorporation at discrete
time points (t1, t2, t2, t3, t4, t7) and integration
for 7 days
Corrected leucine incorporation = average
measurements of leucine incorporation (n =5) –
average leucine incorporation from experimental
controls (n = 5)
Bioavailable N taken up (μg N L-1)=
Corrected leucine incorporation
(nmol of leucine L-1)
÷
N slope (nmol leucine L-1 per μg N L-1)
Bioavailable P
Sub-volume 3
0.2 μm filtration
2 % (v/v) standard inoculum
5 % (v/v) “L16” bacterial growth medium
P-limitation induced
+20000 μg C L-1 as C6H12O6
+2000 μg N L-1 as NH4NO3
7-day incubation at 20 °C in Eppendorf tubes
(5 replicates)
Measurements of leucine incorporation at discrete
time points (t1, t2, t2, t3, t4, t7) and integration
for 7 days
Corrected leucine incorporation = average
measurements of leucine incorporation (n =5) –
average leucine incorporation from experimental
controls (n = 5)
Bioavailable P taken up (μg P L-1)=
Corrected leucine incorporation
(nmol of leucine L-1)
÷
P slope (nmol leucine L-1 per μg P L-1)